Acta Scientiarum

http://www.uem.br/acta
ISSN printed: 1806-2563
ISSN on-line: 1807-8664
Doi: 10.4025/actascitechnol.v35i2.10619

Endocrine disruptors in sludge wastewater treatment plants:

environmental complications
Aldo Pacheco Ferreira
Centro de Estudos da Sade do Trabalhador e Ecologia Humana, Escola Nacional de Sade Pblica Srgio Arouca, Fundao Oswaldo Cruz,
Rua Leopoldo Bulhes, 1480, 21041-210, Manguinhos, Rio de Janeiro, Brazil. E-mail: aldoferreira@ensp.fiocruz.br

ABSTRACT. Concern over exposure to endocrine disruptors (EDs) and impacts on wildlife and human
has gradually increased in importance in recent years. Wastewater systematically receives most if not all of
these chemicals, so a deeper understanding of the fate of EDs in environment is sorely needed. We
evaluated EDs concentration in the affluent and effluent from the Sludge Wastewater Treatment Plants
(SWTPs) Penha and Ilha do Governador. It was used as evaluation parameter the determination of some
target compounds, such as: alkylphenols, 17-estradiol, bisphenol A; complementing with measures of
physical and chemical parameters: temperature, pH, dissolved oxygen, total suspended solids, volatile
suspended solids, and total chemical oxygen demand. Samples were collected along the line of treatment of
each SWTP, and after laboratory procedures, analyzed by LC-MS/MS and ELISA techniques. In some
samples, we verified concentrations of chemical compounds with potential to disrupt the endocrine system
of living organisms. Therefore, the release of this effluent, even within the effluent discharge standards
(BRASIL, 2005) or in condition to be purified by the water course, may lead to physiological changes in the
organisms of the receptor water body.
Keywords: ELISA, LC-MS/MS, water ecophysiology, aquatic ecotoxicology, human sewage, water pollution control.

Desreguladores endcrinos em estaes de tratamento de esgotos: complicaes ao meio

ambiente
RESUMO. A preocupao sobre a exposio aos desreguladores endcrinos (DEs) e impactos sobre a
fauna e os seres humanos, gradativamente, teve aumento em importncia nos ltimos anos. guas residuais
sistematicamente recebem a maioria destes desreguladores, se no todos destes produtos qumicos, assim
maior compreenso sobre o destino de DEs no ambiente extremamente necessria. Foi avaliada a
concentrao de DEs no afluente e no efluente das estaes de tratamento de esgoto (ETEs) da Penha e Ilha
do Governador, Estado do Rio de Janeiro. Foi utilizado como parmetro de avaliao a concentrao dos
seguintes compostos: alquilfenis, 17-estradiol, bisfenol A; complementando com a determinao dos
parmetros fsico-qumicos: Temperatura, pH, Oxignio Dissolvido, Slidos Suspensos Totais, Slidos
Suspensos Volteis e Demanda Qumica de Oxignio total. Foram coletadas amostras simples ao longo da
linha de tratamento de cada ETE estudada e aps procedimentos laboratoriais, analisadas por meio das
tcnicas LC-MS/MS e Elisa. Em algumas amostras, observaram-se concentraes de compostos qumicos
aqui pesquisados com potencial para desregular o sistema endcrino de organismos vivos. Portanto,
conclui-se que o lanamento deste efluente mesmo enquadrado nas normativas de despejo (BRASIL, 2005)
ou apresentando condies de depurao pelo curso hdrico, pode provocar a mudana fisiolgica dos
organismos do ecossistema aqutico receptor.
Palavras-chave: Elisa, LC-MS/MS, ecofisiologia aqutica, ecotoxicologia aqutica, esgoto humano, controle de poluio hdrica.

Introduction
There is a growing concern among scientific
community and public about a set of chemicals in
the environment, which can interact with the
endocrine system and cause adverse effects. Some
studies report the presence of these compounds in
wastewater treatment plants and in surface and
groundwaters, due to the partial removal during the
treatment process, and therefore one part is
Acta Scientiarum. Technology

discharged into receiving water bodies (BILA et al.,

2007; MEYER et al., 1999). These compounds are
usually called as endocrine disruptors (EDs) because
they can affect the health of human as well as of other
organisms (COLBORN et al., 1993). On the other
hand, the exposure to these products may affect all
biological systems, including the reproductive system
and its development (FERREIRA et al., 2006;
RODRIGUEZ-MOZAZ et al., 2004).
Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

308

ED is able to associate with all substance or blend

of exogenous substances and to assume the same
function of a natural hormone in living organisms, or
to inhibit the normal functioning, by changing the
endocrine system and causing adverse effects in
organisms or descendants (TAPIERO et al., 2002).
According to Alves et al. (2007), EDs can lead to
the development of some diseases such as cancer of
the breast, uterus and prostate, abnormal sexual
development, reduced male fertility, increased
incidence of polycystic ovaries, changes in the
thyroid gland, disorders in the ovary, fertilization
and pregnancy. In animals they can disrupt the
production and development of organisms, and may
irreversibly induce female sexual characteristics in
male fish, causing sterility or reduced reproduction
(FOX, 2001).
Several substances are classified as EDs, among
them: natural substances (phytoestrogens), synthetic
chemicals (alkylphenols, pesticides, phthalates,
polychlorinated biphenyls and bisphenol A), natural
estrogens (17- estradiol, estrone and estriol) and
synthetic estrogens (17-ethinylestradiol) (FERREIRA
et al., 2006).
Besides EDs already known, several other new
chemicals are synthetized every year and discharged
into the environment with unknown consequences,
and many of them have potential estrogenic activity.
Indeed, more than 70 chemicals are referred as
potential EDs. However, it is estimated that over
80,000 chemicals produced by men are commonly
used, thus justifying their presence in the effluents
of WTPs, as well as their breakdown products
(BORGES; BARROS JNIOR, 2012; KOLPIN
et al., 2002; LEE et al., 2005).
Polyethoxylated alkylphenols (PEAs) are widely
used in domestic and commercial applications, being
used as emulsifiers in industrial and domestic
cleaning products (KOLPIN et al., 2002). During
the treatment of urban and industrial wastewaters,
PEAs are successively degraded to less biodegradable
substances, e.g., the nonylphenol (NP) and
octylphenol (OP), in order to be discharged into the
aquatic system (CHENG et al., 2006).
In general, WTPs receive fresh sewage and
subject it to a series of physical, chemical and
biological processes aiming to remove undesirable
substances from contaminated water, allowing its
return to the environment with more suitable
sanitary characteristics (BENTO et al., 2005;
FALCIONI et al., 2005; FERREIRA et al., 2008).
There is lacking information available relative to the
effect of high discharges and their potential toxicity,
especially in aquatic systems. Moreover, little
information on the removal of EDs in WTPs
Acta Scientiarum. Technology

Ferreira

prevents the estimation of material balances. In this

way, several bioassays and analytical techniques have
been employed to identify and examine the activity
of several potentially estrogenic compounds
(MARA, 2003).
17- estradiol is responsible for the formation of
female characteristics and 17-ethinylestradiol is the
main synthetic estrogen found in contraceptive pills
and applied in hormone replacement therapies,
these substances have high estrogenic potential and
are classified as the main responsible for endocrine
changes in organisms present in surface waters
(OLEA-SERRANO et al., 2002). These compounds
are detected in effluents of WTPs since the
treatment process does not completely remove them
from received wastewater (MORAES et al., 2008).
In this study, we analyzed the effluent treated in
the SWTPs Penha and Ilha do Governador, in order
to evaluate the yield and quality of the processes,
intending thus to subsidize future projects of the
SWTPs, allowing the improvement of performance
and monitoring, resulting in better control of the
process and the reduction of potential impacts
caused on the environment by EDs.
Endocrine disruptors: framework and definition

The first studies that related the effect of some

chemicals in animals emerged during the 1950s and
1960s by Rachel Carson who associated the impacts
of the Dichloro-Diphenyl-Trichloroethane (DDT)
in the environment with those in animals
(CARSON, 1962). However, public concern over
pollution problems only emerged with the
publication of Our stolen future: Are we
threatening our fertility, intelligence and survival?.
In recent decades several studies have been
conducted to relate properties of endocrine
disruption of some compounds (natural and
produced by men) mainly at the level of hormonal
activity (SUMPTER; JOHNSON, 2005). There is
no agreement on EDs and methods to determine,
measure, analyze and test them in various types of
organisms.
The appropriate choice and development to
identify and characterize chemicals that can disrupt
the endocrine system are very complicated. For
example, the receiver of such compounds does not
follow the standard rules for toxicity by the
endogenous levels of hormones present in the body.
The EDs may operate through pathways different
from the other compounds producting distinct
responses even at very low concentrations. On the
other hand, there is a great number of available tests
validated and based on a wide array of mechanisms
completely different (WAISSMANN, 2002).
Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

Estrogen in SWTPs

By detecting the significant levels of compounds

able of endocrine disruption in the environment
emerged the need to define EDs. Nevertheless, all
the attempts to define EDs include the concept of
deregulation or disruption any effect (adverse or
not) on the endocrine system.
This is related to small biochemical changes or
small changes at cell level that can lead to great
damages in the long term. Actually changes in
endocrine system occur systematically by natural
environmental factors, and frequently small
disturbances from anthropogenic compounds
released in the environment do not even cause any
pathology. The question is to what extent or to
which concentration of EDs each individual exposed
can adapt without any pathology. The consequences
of the exposure can be displayed not only at
individual level, but also at level of its offspring, i.e.,
there may be changes and effects at population level
(WANG et al., 2005).
Identification, quantification and analysis:
presence of ED in the environment
According to several authors there are between
50 and 70 different chemicals identified as being
possible EDs (ISHIBASHI et al., 2001;
MATTHIESEN; GIBBS, 1998). However, as more
chemicals are tested, the number with estrogenic
responses also increases, i.e., the list of compounds
considered EDs is far from complete, and has not
stopped increasing with the development of
technology and new tests have arisen.
One of the most common ways of exposure to
EDs is through the contact with contaminated water
of rivers, bays, groundwaters etc. (FERREIRA et al.,
2006). The EDs can reach the water by several ways:
(a) punctual sources effluents of wastewater
treatment plants, effluents of industries, agricultural
activities, leachates etc; and (b) diffuse sources soil
infiltration of compounds used in the agriculture
and industry until reaching groundwater, recharge
of aquifers with contaminated water, septic tanks,
spread of sludge from wastewater treatment, etc
(Figure 1).
The EDs similarly to other pollutants have a
great variety of sources. These sources can have
implications to the environment (due to their
accumulation) and adverse effects to humans and
other living organisms (BIRKETT; LESTER, 2003).
Surface waters are particularly susceptible to
contamination by EDs, given the proximity to
pollution sources and the low dilution factor, which
originates a high degree of exposure of all organisms
that come into contact. Furthermore, the treatment
performed in the WTPs is not sufficient (FERREIRA
et al., 2006). This because, in some cases, the
Acta Scientiarum. Technology

309

degradation products of metabolism in the WTPs have

a greater estrogenic potential. One example is the
APEOs that degrade into NP. These compounds have
been largely used in the last 50 years in a great array of
domestic and commercial applications (TSUDA et al.,
2000), as emulsifiers in domestic and industrial
cleaning products (NICHOLS et al., 2001). During
the treatment of urban and industrial wastewater,
APEOs are successively degraded into less
biodegradable forms, such as for example, NP and OP,
which are thus released into aquatic ecosystems
(MAGUIRE, 1999).

Figure 1. Potential routes of exposure to human hormones in the

environment.

Moreover, alkylphenols (NP, OP) induce the

production of vitellogenin in male individuals of
several fish species (SCHWAIGER et al., 2000). Other
estrogenic contaminants found in the water include
ethinylestradiol,
phytoestrogens,
organochlorine
compounds, among others vulnerable to disrupt the
endocrine system of fish and other organisms.
The 17 -estradiol is the main estrogen present in
vertebrates, and in female fish it regulates the
development and maintenance of gonads and somatic
sexual characteristics and has a key role in the
vitellogenesis (ASHFIELD et al., 1998). This justifies
the great importance of studying of its concentration
and the effects at the level of the endocrine system of
fish. Another large group of EDs are the heavy metals,
such as cadmium, lead and mercury, which despite not
interfering with hormone activity, under high
concentrations are toxic to gonadal cells of fish
Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

310

Ferreira

(NELSON et al., 2007). Some of these substances

were banned or no longer produced, but still found in
the environment. In the Table 1 are listed the main
substances known as EDs.
Among the EDs, natural and synthetic estrogens
have called attention of some researchers, being
found in the environment at concentrations in the
range of g L-1 and ng L-1. Likewise, have been
found in cosmetics and anabolics used in animal
feed, and also considered EDs (ALVES et al., 2007).
In this way, we can mention polychlorinated
biphenyls (PCBs), bisphenol A (BPA), dioxins
(PCDDs), furans (PCDFs), DDT and alkylphenol
polyethoxylates compounds, such as OP and NP.
BPA is an ingredient commonly found in several
products for dental treatments, as also in internal
coatings of food packages, which is a facilitator for
human contamination (STUMPF et al., 1999).
Natural estrogens, estrone and 17-estradiol are
naturally and daily excreted in the urine of women,
female animals and men, and thus discharged in
domestic sewage, as well as the 17- ethinylestradiol
is a synthetic estrogen used in contraceptive pills
(BILA et al., 2007). Table 2 presents the daily
excretion of estrogens by human.
Focusing on the study and evaluation of the
estrogenic potential in the treatment steps of the
SWTPs Penha and Ilha do Governador, it was
determined some EDs, such as: alkylphenols
(nonylphenol and octylphenol), 17 -estradiol and
bisphenol A. Samples were taken along the line of
treatment of each SWTPs, which after processed were
analyzed by LC-MS/MS and ELISA techniques.
Material and methods
Characterizing the STP

The SWTPs Penha and Ilha do Governador

are submitted to the following operational

parameters: pH, total suspended solids (TSS),

fixed suspended solids (FSS), volatile suspended
solids (VSS), chemical oxygen demand (COD),
biochemical oxygen demand (BOD), total
Kjeldahl nitrogen, ammonia nitrogen, nitrate,
nitrite and phosphorus.
SWTP Penha

The SWTP Penha operates with biofilters and

activated sludge, treating a flow of around 1,600 L s-1.
The pre-treatment starts by medium and fine
screening, with removal of solids, followed by
removal of sands, oils, and greaset. Greases are
incinerated. The primary treatment consists of an
accelerated lamellar settling in four tanks, with
optional physical and chemical treatment, and of
three tanks of equalization/homogenization. The
secondary biological treatment is performed by
means of a continuous-flow activated sludge system
with conventional aeration in six aerated tanks by
surface aeration, followed by a secondary lamellar
settling, in 12 settlers of rectangular plant, with
biological sludge recirculation.
STP Ilha do Governador

The SWTP Ilha do Governador operates with

activated sludge, treating a flow of around 525 L s-1.
The treatment process works with the steps: the
elevated influent pass through a screening formed
by grids a coarse and a medium, responsible for
removing coarse solids, followed by the removal of
oils,
greases
and
sands,
in
two
grit
chambers/degreasers. The resulting byproducts of
this pre-treatment are later deposited in controlled
landfill. The influent is then fed to a
homogenization tank e with 4,000 m3 working
volume, whose function is to regularize peaks of
pollutant load.

Table 1. Main substances of EDs.

Steroids
17- ethinylestradiol
17-estradiol
Estrone
Mestranol
Diethylstilbestrol (DES)

Alkylphenols

Polyaromatic compounds

Nonylphenol
Ethoxylated nonylphenol
Octylphenol

Polychlorinated biphenyls (PCB)

Flame retardants
Polyaromatic hydrocarbons (PAH

Oxygenated organic
compounds
Phthalates
Bisphenol A

Pesticides
Atrazine
Linuron
Hexachlorobenzene
Pentachlorophenol (PCP)

Source: Bila et al. (2007).

Table 2. Daily excretion (g) per capita of estrogens by humans.

Men
Women of childbearing age (10 - 49 years)
Menopausal women (above 49 years)
Pregnant women
Women taking contraceptive

17-estradiol
1.6
3.5
2.3
259
-

Estrone
3.9
8
4
600
-

Estriol
1.5
4.8
1
6000
-

17-ethinylestradiol
35

Source: Johnson et al. (2000).

Acta Scientiarum. Technology

Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

Estrogen in SWTPs

The subsequent primary treatment consists of a

preliminary step of physical and chemical treatment,
with application of aluminum sulfate, hydrated lime
and a polyelectrolyte on two lines of
coagulation/flocculation tanks, with primary settling
in two tanks of circular plant with bottom and
surface scraper. The primary effluent is then fed to
the secondary treatment, which is performed by
means of activated sludge system, in six aeration
tanks provided of two surface aerators each and
sludge recirculation tank. The next step is the
secondary settling in two settlers of circular plant,
with bottom and surface scraper.
Sampling
Samplings were carried out along the line of
treatment of the SWTPs Ilha do Governador and
Penha. The sites sampled were: (1) influent, (2) postpreliminary treatment, (3) post-primary treatment and
(4) final effluent.
Samples were taken in triplicate and stored in
amber vials of 250 mL. During the 12h scheduled for
sample collection, which occurred between 8 and 20h,
it was gathered eight samples per site during the
collection period. The collections in the SWTPs were
performed in March 2010, and the results express the
consolidated obtained from each analyzed site.
Laboratory analyses

After collection, samples were taken to the

laboratory in chilled bags, filtered through a mesh of
230 m to retain larger particles, and then through
glass fiber filters Macherey-Nagel, MN GF -3,
porosity of 0.60 m. The methodology for the pretreatment of samples and for extraction of different
compounds was developed for analysis in Elisa
(Indirect immunosorbent assay) by Takeda Chemical

311

Industries Ltd. and for analysis in LC-MS/MS (Liquid

chromatography-mass spectrometry) (PETROVIC
et al., 2002).
In the analysis through Elisa, the extraction of BPA,
17Estradiol and APE was undertaken using Oasis
HLB cartridge 3 cc 60 mg-1 30 m 100 box-1
(WAT094226, Waters), being eluted with 2 x 5.0 mL
CH3OH at 10% for the BPA and APE, and with 2 x 5.0
mL CH2Cl2 (100%) for the 17Estradiol. Afterwards,
samples were concentrated to 2 mL with liquid nitrogen.
The processing scheme is shown in the Figure 2.
The Elisa assay is based on a reaction of
competition between a specific monoclonal antibody
and the compound under analysis. In this reaction, the
surface of the wells is coated with a protein
(monoclonal antibody) that binds exclusively to the
ED (antigen) in analysis. The ED, under study, present
in the samples analyzed and the conjugate (antigen
enzyme), (ED marked with an enzyme that changes
color in reaction) were previously mixed and added to
the microplate wells, taking place thus a competition
for the limited number of bonds to the antibody.
Later, the excess of conjugate and the unreacted ED
were removed from the wells using a wash solution
(PBS/Tween). Then each well received a chromogenic
substrate, in order to develop color under the presence
of the enzyme conjugate. The amount of this bound to
the antibody determined the color intensity, which in
turn was measured by absorbance using an Elisa reader
(BenchMark, Biorad).
The quantitative analysis was done by selected ion
monitoring (SIM), using an external calibration.
Calibration curves were built by a linear regression.
The confirmation of the identity of the compounds in
the effluent was performed by means of scan mode, by
comparing the retention time and the mass spectrum
of the compound analyzed with the respective
standard.

Figure 2. Scheme of processing and analysis of the samples.

Acta Scientiarum. Technology

Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

312

Ferreira

Analyses by LC-MS/MS were accomplished in

Agilent 1100 system, with automatic sampler coupled
to a mass spectrophotometer MDS/SCIEX API2000
(Turbo Ion Spray), with a reverse phase column C18 (5 m, 250 x 4 mm - LiChrospher 100 RP-18)
and a pre-column (4 x 4 mm, 5 m - Merck,
Darmstadt, Germany). The injection volume was
adjusted to 25 L at a flow rate of 1 mL min.-1.
Physical and chemical parameters were determined
as follow: pH readings were made in situ using portable
equipment (Digimed DM/2P). The analyses of TSS,
FSS, VSS, COD, BOD, total Kjeldahl N, ammonia
N, nitrate, nitrite and phosphorus were performed
according to Standard Methods for the Examination of
Water and Wastewater (AWWA, 1995).
Statistical analysis

Statistical analyses were run using the software

Origin 7.5 (OriginLab Corporation).
Results and discussion
The results obtained for the EDs, from the
effluents collected along the line of treatment of the
SWTPs Penha and Ilha do Governador are
presented in Tables 3 and 4, with analyses using the

methodology LC-MS/MS; and in Tables 5 and 6,

with analyses using the methodology Elisa.
The results obtained by several authors for
17-estradiol in effluents of WTPs in Germany
(5.2 ng L-1), England (1.6 ng L-1) Canada (2 ng L-1),
USA (6.5 ng L-1) and Japan (0.5 ng L-1),
(DORABAWILA;
GUPTA,
2005;
KUCH;
BALLSCHMITER, 2001; LEE et al., 2005) were
within the range of 0.5 6.5 ng L-1. Our results using
the LC-MS/MS method for the same compound in
the effluent of the SWTP Penha were in the average,
with the range of 0.18 0.77 ng L-1 (Xmin= 0.09 and
Xmax= 1.11) and of the SWTP Ilha do Governador
with the range of 0.41 3.23 ng L-1 (Xmin= 0.21 and
Xmax= 5.67). However, when the 17-estradiol was
examined by Elisa in the effluent of the SWTP Penha it
was detected the range of 0.66 2.91 ng L-1 (Xmin= 0.43
and Xmax= 3.77), and in the SWTP Ilha do
Governador, the range of 0.41 3.23 ng L-1 (Xmin= 0.21
and Xmax= 5.67). Considering that the abovementioned
studies had not used the Elisa method, our results
indicate a significant difference of method, showing
better results that pointed values closer to the results
observed in Germany and USA, and higher than in
England, Canada and Japan.

Table 3. Analyses of concentrations of EDs by the LC-MS/MS method in the effluent of the SWTP Penha.
Sites
1
2
3
4
1
2
3
4
1
2
3
4

EDs
17 estradiol
(ng L-1)
BPA
(g L-1)
APEOs
(g L-1)

X
0.77
0.65
0.92
0.18
4.44
2.12
2.72
1.05
52.76
69.94
47.08
10.34

Sd(yEr)
0.20708
0.19241
0.19456
0.04422
0.91526
0.5646
0.6904
0.31802
7.47733
6.42292
6.93132
2.10333

Se(yEr)
0.07322
0.06803
0.06879
0.01563
0.32359
0.19962
0.24409
0.11244
2.64363
2.27085
2.45059
0.74364

P25
0.61
0.48
0.72
0.16
3.37
1.86
2.14
0.89
45.86
63.67
42.33
8.45

SWTP Penha
LC-MS/MS method
P75
P95
Xmin
0.88
1.11
0.56
0.68
1.04
0.46
1.07
1.19
0.69
0.21
0.23
0.09
5.14
5.31
2.78
2.54
2.64
0.98
2.81
4.05
1.79
1.04
1.79
0.71
59.27 63.84 44.89
76.12 78.25 62.77
51.19 57.48 37.38
11.58 13.86
8.19

Xmax
1.11
1.04
1.19
0.23
5.31
2.64
4.05
1.79
63.84
78.25
57.48
13.86

R
0.55
0.58
0.5
0.14
2.53
1.66
2.26
1.08
18.95
15.48
20.1
5.67

Median
0.7
0.63
0.925
0.185
4.68
2.21
2.705
0.99
49.395
69.29
45.49
9.78

Var
0.04288
0.03702
0.03786
0.00196
0.8377
0.31877
0.47666
0.10114
55.91039
41.25396
48.04316
4.42401

CoefVar
0.26764
0.29264
0.21006
0.24397
0.20608
0.26585
0.25382
0.30216
0.14172
0.09182
0.1472
0.20339

X-arithmetic mean; Sd- standard deviation; Se- standard error; P25 Percentile 25; P75 Percentile 75; P95 Percentile 95; Xmin- minimum; Xmax-maximum; R Range; Var Variance; CoefVarcoefficient of variation (%).

Table 4. Analyses of concentrations of EDs by the LC-MS/MS method in the effluent of the SWTP Ilha do Governador.
Sites
1
2
3
4
1
2
3
4
1
2
3
4

EDs

17 estradiol
(ng L-1)
BPA
(g L-1)
APEOs
(g L-1)

X
0.96
1.02
1.11
0.26
1.77
1.94
0.78
0.41
32.15
25.55
29.08
5.15

Sd(yEr)
0.22605
0.26259
0.25182
0.06413
0.53973
0.76859
0.22564
0.15682
10.79091
8.94666
10.20934
1.91314

Se(yEr)
0.07992
0.09284
0.08903
0.02267
0.19082
0.27174
0.07978
0.05544
3.81516
3.16312
3.60955
0.6764

P25
0.71
0.67
0.91
0.22
1.28
1.39
0.6
0.23
21.69
16.57
18.78
3.17

SWTP Ilha do Governador

LC-MS/MS method
P75
P95
Xmin
Xmax
1.19
1.24
0.68
1.24
1.17
1.33
0.64
1.33
1.2
1.58
0.82
1.58
0.31
0.34
0.15
0.34
1.96
2.77
1.26
2.77
2.31
3.08
0.66
3.08
0.86
1.17
0.46
1.17
0.51
0.67
0.22
0.67
38.69 47.23 18.64
47.23
31.31 38.45 15.33
38.45
35.62 43.81 17.43
43.81
6.44
7.91
3.03
7.91

R
0.56
0.69
0.76
0.19
1.51
2.42
0.71
0.45
28.59
23.12
26.38
4.88

Median
0.92
1.12
1.06
0.265
1.575
1.815
0.77
0.39
31.44
24.895
28.33
5.1

Var
0.0511
0.06896
0.06341
0.00411
0.29131
0.59074
0.05091
0.02459
116.44367
80.0428
104.23056
3.66009

CoefVar
0.23516
0.25713
0.22661
0.24314
0.30429
0.39567
0.28928
0.38017
0.33563
0.35011
0.35105
0.37148

X-arithmetic mean; Sd- standard deviation; Se- standard error; P25 Percentile 25; P75 Percentile 75; P95 Percentile 95; Xmin- minimum; Xmax-maximum; R Range; Var
Variance; CoefVar- coefficient of variation (%).

Acta Scientiarum. Technology

Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

Estrogen in SWTPs

313

Table 5. Analyses of concentrations of EDs by the Elisa method in the effluent of the SWTP Penha.
Sites
1
2
3
4
1
2
3
4
1
2
3
4

EDs

17 estradiol
(ng L-1)
BPA
(g L-1)
APEOs
(g L-1)

X
2.91
2.57
2.13
0.66
5.13
2.74
2.36
1.34
65.32
61.91
51.12
13.11

Sd(yEr)
0.67324
0.59464
0.58674
0.22335
1.96571
1.03593
0.91872
0.51826
25.63814
24.75357
19.55092
6.34904

Se(yEr)
0.23803
0.21024
0.20745
0.07897
0.69498
0.36626
0.32482
0.18323
9.06445
8.75171
6.9123
2.24473

P25
2.17
1.92
1.86
0.43
2.88
1.58
1.32
0.75
36.42
34.22
28.5
7.31

P75
3.37
2.96
2.54
0.68
6.38
3.39
2.92
1.66
80.86
76.54
63.21
15.21

SWTP Penha
Elisa method
P95
Xmin
Xmax
3.77
2.02
3.77
3.32
1.77
3.32
2.76
0.95
2.76
1.09
0.43
1.09
8.35
2.68
8.35
4.43
1.41
4.43
3.89
1.21
3.89
2.21
0.71
2.21
106.76 31.62 106.76
102.49 30.09 102.49
84.03
27.26
84.03
24.29
3.99
24.29

R
1.75
1.55
1.81
0.66
5.67
3.02
2.68
1.5
75.14
72.4
56.77
20.3

Median
3.07
2.71
2.21
0.625
4.85
2.585
2.235
1.255
62.78
58.43
48.33
12.395

Var
0.45326
0.3536
0.34427
0.04989
3.86403
1.07316
0.84406
0.26859
657.31427
612.73913
382.23864
40.31037

CoefVar
0.23136
0.23138
0.27531
0.33841
0.38318
0.37825
0.38867
0.38604
0.39249
0.39984
0.38246
0.48429

X-arithmetic mean; Sd- standard deviation; Se- standard error; P25 Percentile 25; P75 Percentile 75; P95 Percentile 95; Xmin- minimum; Xmax-maximum; R Range; Var
Variance; CoefVar- coefficient of variation (%).

Table 6. Analyses of concentrations of EDs by the Elisa method in the effluent of the SWTP Ilha do Governador.
Sites
1
2
3
4
1
2
3
4
1
2
3
4

EDs

17 estradiol
(ng L-1)
BPA
(g L-1)
APEOs
(g L-1)

X
3.23
2.76
1.45
0.41
3.32
2.29
1.38
0.72
45.10
40.13
33.45
10.17

Sd(yEr)
1.41932
1.05107
0.41074
0.10849
1.45969
1.08775
0.61557
0.31842
19.56435
18.21837
14.94038
4.73662

Se(yEr)
0.5018
0.37161
0.14522
0.03836
0.51608
0.38458
0.21764
0.11258
6.91704
6.44117
5.28222
1.67465

P25
1.83
1.54
1.23
0.35
1.88
1.37
0.78
0.41
28.19
22.81
18.31
5.76

SWTP Ilha do Governador

Elisa method
P75
P95
Xmin
Xmax
3.97
5.67
1.22
5.67
3.41
4.49
1.47
4.49
1.73
1.9
0.64
1.9
0.49
0.54
0.21
0.54
4.08
5.81
1.23
5.81
2.81
4.03
0.56
4.03
1.69
2.45
0.51
2.45
0.89
1.27
0.27
1.27
55.55 78.34 15.19 78.34
49.47 71.15 13.22 71.15
41.23 58.89 12.17 58.89
12.53 18.33
3.13
18.33

R
4.45
3.02
1.26
0.33
4.58
3.47
1.94
1.00
63.15
57.93
46.72
15.2

Median
3.11
2.61
1.48
0.42
3.195
2.2
1.325
0.695
43.52
38.75
32.295
9.805

Var
2.01446
1.10474
0.16871
0.01177
2.1307
1.18319
0.37893
0.10139
382.76391
331.90908
223.21505
22.43554

CoefVar
0.43874
0.38082
0.28376
0.26221
0.43983
0.47552
0.44566
0.44072
0.43375
0.45403
0.44668
0.46552

X-arithmetic mean; Sd- standard deviation; Se- standard error; P25 Percentile 25; P75 Percentile 75; P95 Percentile 95; Xmin- minimum; Xmax-maximum; R Range; Var
Variance; CoefVar- coefficient of variation (%).

In relation to BPA, the obtained results with the

LC-MS/MS method in the effluent of the SWTP
Penha were in the average with the range of 1.05 4.44
g L-1 (Xmin= 0.71 and Xmax= 5.31) and in the SWTP
Ilha do Governador within the range of 0.41 1.77 g
L-1 (Xmin= 0.22 and Xmax= 2.77). With the Elisa
method in the effluent of the SWTP Penha, we
detected a range of 1.34 5.13 g L-1 (Xmin= 0.75 and
Xmax= 8.35) and in the SWTP Ilha do Governador, the
range of 0.72 3.32 g L-1 (Xmin= 0.27 and
Xmax= 5.81). Significantly different results were also
verified between methods, indicating a better result
using the Elisa method. The BPA is a substance used in
industries as a component of epoxy resins. In the
environment, this compound has a high persistence.
Values determined in the effluents of both SWTPs are
within the interval registered by Heisterkamp et al.
(2004), which detected levels between 0.085 and 28 g
L-1, but the final effluent had on average 6.09 g L-1,
higher than found in the present study.
Regarding the APEOs (NP and OP), LC-MS/MS
method results in the effluent of the SWTP Penha
were on average within the range of 10.34 52.76 g
L-1 (Xmin= 8.19 and Xmax= 63.84) and in the SWTP
Ilha do Governador, between 5.15 32.15 g L-1
(Xmin= 3.03 and Xmax= 47.23). Using the Elisa method,
Acta Scientiarum. Technology

the range of 13.11 65.32 g L-1 (Xmin= 3.99 and

Xmax= 106.76) was found in the effluent of the SWTP
Penha, and the range of 10.17 45.10 g L-1
(Xmin= 3.13 and Xmax= 78.34) in the SWTP Ilha do
Governador. In the last 50 years, the APEOs were
largely used in industrial, commercial and domestic
applications, with an annual production of 350,000
ton., in the USA, Europe and Japan, explaining their
presence in the SWTPs (NICHOLS et al., 2001). This
justifies the current growing concern about the
indiscriminate use of these chemicals, especially due to
the relative stability of some metabolits and
degradation of products containing APEOs (TSUDA
et al., 2000). Results achieved with the Elisa method
were better than the LC-MS/MS.
All analyses pointed out a higher sensitivity to the
Elisa method in detecting EDs. These two techniques
were chosen to be tested given their relative
accessibility and high reproducibility, subsidizing thus
the research on EDs and contributing to make more
reliable the conclusions made with the study.
Nevertheless, in all cases regardless of the method
(Elisa or LC-MS/MS) the selected EDs were present in
the effluents of both SWTPs, with varied
concentrations, which are released into the Guanabara
Bay, with levels with potential physiological effects on
Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

314

Ferreira

animal life (COLBORN et al., 1993; FERREIRA et al.,

2006). But taking into account the dilution level in this
bay, it is expected that the effects on the organisms are
not relevant and/or immediate.
The Table 7 and Table 8 present the values of
physical and chemical parameters of treatment control
in the SWTPs Penha and Ilha do Governador,
respectively. The behavior of these parameters was
consistent with that expected; indicating the high
efficiency of the process in the SWTPs. Importantly,
the results of key parameters meet the requirement of
Conama 357 (BRASIL, 2005), such as pH within the
range of 5 and 9, and up to 60 mg L-1 of BOD and
ammonia N amoniacal up to 20 mg L-1.
Moreover, despite the efficiency of the SWTPs,
there is a need for further procedures at the end of the
process, aiming to reduce EDs to safe levels. Literature
data on the removal efficiency of estrogens in WTPs
reinforce the need for subsequent treatments in the
effluent (LOPES et al., 2008). According to Andersen
et al. (2003) in Arkansas (USA) the removal of EDs
was more effective in the processes for nutrient
removal, i.e., the tertiary treatment responsible for
removing nitrogen and phosphorus. In this case, the
sludge presented a retention time of 11-13 days with
the growth of microorganisms able to degrade
estrogens, which is not observed in conventional
methods with the sludge retention time shorter than
four days.

This study showed that concentrations of the

studied compounds in the effluents of the SWTPs have
been at worrying levels relative to levels of EDs in
effluents of these countries, reflecting the need for
research and implementation of a greater control in the
WTPs, strengthening basic sanitation of the country,
minimizing thus significant impacts to the
environment.
Analytical techniques used herein allowed
comparing values of EDs in the different process
steps of the SWTPs. Data obtained with Elisa had
high detectability and selectivity in relation to the
LC-MS/MS method, with a greater sensitivity to the
EDs, and therefore presented better detection limits.
Parameters for validation observed for the procedure
increased the reliability of the results, once there is
no official method to simultaneously determine
these chemicals. This procedure could also base
monitoring studies on these compounds in
effluents.
A suitable policy to reduce the threat of
chemicals with potential to affect the hormone
system requires a control on the availability and
guidelines for using pesticides like endosulfan and
methoxychlor, fungicides such as vinclozolin,
herbicide like atrazine, alkylphenols, phthalates and
BPA. In order to prevent the generation of dioxin is
essential the gradual elimination of PVC,
tetrachlorethylene and chlorinated pesticides.

Table 7. Physical and chemical parameters presented in the sampling sites of the SWTP Penha.
SWTP Penha
Parameter
pH
TSS (mg L-1)
FSS (mg L-1)
VSS (mg L-1)
COD (mg O2 L-1)
BOD (mg O2 L-1)
Total Kjeldahl N (mg N L-1)
Ammonia N (mg N-NH3 L-1)
Nitrate (mg NO3 L-1)
Nitrite (mg NO2 L-1)
Total phosphorus (mg P L-1)

1
6.62
4265.2
964.5
3198.3
25460
5745
333.7
84.8
188.6
123.2
4.41

2
6.78
1910.7
267.7
1625.8
18636
3567
165.9
61.6
22.1
16.7
4.78

Sampling sites
3
6.45
108.6
132.8
121.8
254
566
13.1
21.4
3.5
1.7
2.67

4
6.87
92.1
35.9
41.0
233
58
11.8
4.5
1.7
0.5
2.34

Efficiency (%)
97.8
96.27
98.71
99.08
98.99
96.46
94.6
90.98
95.94
46.93

Table 8. Physical and chemical parameters presented in the sampling sites of the SWTP Ilha do Governador.
SWTP Ilha do Governador
Parameter
pH
TSS (mg L-1)
FSS (mg L-1)
VSS (mg L-1)
COD (mg O2 L-1)
BOD (mg O2 L-1)
Total Kjeldahl N (mg N L-1)
Ammonia N (mg N-NH3 L-1)
Nitrate (mg NO3 L-1)
Nitrite (mg NO2 L-1)
Total phosphorus (mg P L-1)

Acta Scientiarum. Technology

1
6.34
5642.7
678.9
2433.7
18453
5997
452.3
112.5
231.4
98.2
3.77

2
6.43
1455.3
199.4
1322
12642
6622
177.6
48.7
77.2
22.6
2.15

Sampling sites
3
6.77
67.9
111.1
87.3
278
475
16.7
19.8
4.1
2.4
1.87

4
6.93
48.2
24.4
22.2
151
44
10.2
7.6
1.5
0.3
0.87

Efficiency (%)
99.1
96.4
99.08
99.18
99.26
97.74
93.24
93.51
96.94
76.92

Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

Estrogen in SWTPs

Conclusion
This study adds knowledge to a little studied
topic in the country, which produced data that
allowed understanding the treatment and presence
of EDs in two important SWTPs. Given the
potential to disrupt the endocrine system it is
necessary to continue the researches on
methodologies for detection and on the action of
EDs in the environment, and on the reproductive
system of the fauna in aquatic systems. Something
positive, Brazil already has public policies that place
the country on par with European countries and
USA, referring the control of EDs in SWTPs.
Acknowledgements
To Faperj, by financial support.
References
ALVES, C.; FLORES, L. C.; CERQUEIRA, T. S.;
TORALLES, M. B. P. Exposio ambiental a interferentes
endcrinos com actividade estrognica e sua associao
com distrbios puberais em crianas. Cadernos de
Sade Pblica, v. 23, n. 5, p. 1005-1014, 2007.
ANDERSEN, H.; SIEGRIST, H.; HALLINGSORENSEN, B.; TERNES, T. A. Fate of estrogens in a
municipal sewage treatment plant. Environmental
Science and Technology, v. 37, n. 18, p. 4021-4026,
2003.
ASHFIELD, A. L.; POTTINGER, T. G.; SUMPTER, J.
P. Exposure of female juvenile rainbow trout to
alkylphenolic compounds results in modifications to
growth and ovosomatic index. Environmental
Toxicology and Chemistry, v. 17, n. 3, p. 679-686,
1998.
AWWA-American Water Works Association. Standard
methods for the examination of water and
wastewater. 19th ed. Washington, D.C.: APHA, 1995.
BENTO, A. P.; SEZERINO, P. H.; PHILIPPI, L. S.;
REGINATTO, V.; LAPOLLI, F. R. Caracterizao da
microfauna em estao de tratamento de esgotos do tipo
lodos ativados: um instrumento de avaliao e controle do
processo. Engenharia Sanitria e Ambiental, v. 10,
n. 4, p. 329-338, 2005.
BILA, D. M.; MONTALVO, A. F.; AZEVEDO, D. A.;
DEZOTTI, M. Estrogenic activity removal of 17Estradiol by ozonation and identification of by-products.
Chemosphere, v. 69, n. 5, p. 736-746, 2007.
BIRKETT, J. W.; LESTER, J. N. Endocrine disrupter
in wastewater and sludge treatment process. Boca
Raton: CRC Press LLC, 2003.
BORGES, M. E. E.; BARROS JNIOR, C. Physicchemical evaluation of leach and water from the borba
gato streamlet within the catchment area of the urban
waste landfill of maring, paran state, Brazil. Acta
Scientiarum. Technology, v. 34, n. 1, p. 41-51, 2012.
Acta Scientiarum. Technology

315

BRASIL. Ministrio do Meio Ambiente, Conselho

Nacional do Meio Ambiente. Resoluo n. 357, de 17 de
maro de 2005. Dirio Oficial da Unio, seo 1, 18 de
Maro de 2005. Available from: <http://www.mma.
gov.br>. Access on: Aug. 23, 2011.
CARSON, R. Silent Spring. Boston: Houghton Mifflin
Company, 1962.
CHENG, C. Y.; WU, C. Y.; WANG, W. H.; DING, W. H.
Determination and distribution characteristics of degradation
products of nonylphenol polyethoxylates in the rivers of
Taiwan. Chemosphere, v. 65, n. 11, p. 2275- 2281, 2006.
COLBORN, T.; VON-SAAL, F.; SOTO, A.
Developmental effects of endocrine-disrupting chemicals
in wildlife and humans. Environmental Health
Perspectives, v. 101, n. 5, p. 378-384, 1993.
DORABAWILA, N.; GUPTA, G. Endocrine disrupter-estradiol--in Chesapeake Bay tributaries. Journal of
Hazardous Materials, v. 120, n. 1-3, p. 67-71, 2005.
FALCIONI, T.; MANTI, A.; BOI, P.; CANONICO, B.;
BALSAMO, M.; PAPA, S. Enumeration of activated
sludge bacteria in a wastewater treatment plant. Journal
of Biological Regulators and Homeostatic Agents,
v. 19, n. 3-4, p. 176-179, 2005.
FERREIRA, A. P.; ANDRADE-RIBEIRO, A. L.; CUNHA,
C. L. N.; KLING, A. S. M. Disruptores endocrinos: potencial
problema para la salud pblica y medio ambiente. Revista
Biomdica, v. 17, n. 2, p. 146-150, 2006.
FERREIRA, A. P.; CUNHA, C. L. N.; ROQUE, O. C.
C. N. Avaliao da microfauna no efluente final para
monitoramento da qualidade ambiental em estaes de
tratamento de esgotos do tipo lodos ativados. Gaia
Scientia, v. 1, n. 2, p. 51-58, 2008.
FOX, G. A. Wildlife as sentinels of human health effects
in the Great Lakes - St. Lawrence basin. Environmental
Health Perspectives, v. 109, n. 6, p. 853-861, 2001.
HEISTERKAMP, I.; GANDRASS, J.; RUCK, W.
Bioassaydirected Chemical analysis utilizing LcMs: A
tool for identifying estrogenic compounds in water
samples? Analytical and Bioanalytical Chemistry,
v. 378, n. 3, p. 709-715, 2004.
ISHIBASHI, H.; TACHIBANA, K.; TSUCHIMOTO,
M.; SOYANO, K.; ISHIBASHI, Y.; NAGAE, M.;
KOHRA, S.; TAKAO, Y.; TOMINAGA, N.; ARIZONO,
K. In vivo testing system for determining the estrogenic
activity of endocrine-disrupting chemicals (EDCs) in
goldfish (Carassius auratus). Journal of Health Science,
v. 47, n. 2, p. 213-218, 2001.
JOHNSON, A. C.; BELFROID, A.; DI CORCIA, A.
Estimating steroid oestrogen inputs into activated sludge
treatment works and observations on their removal from
the effluent. The Science of the Total Environment,
v. 256, n. 2-3, p. 163-173, 2000.
KOLPIN, D. W.; FURLONG, E. T.; MEYER, M. T.;
THURMAN, E. M.; ZAUGG, S. D.; BARBER, L. B.;
BUXTON, H. T. Pharmaceuticals, hormones, and other
organic wastewater contaminants in U.S. streams, 1999-2000:
A national reconnaissance. Environmental Science and
Technology, v. 36, n. 6, p. 1202-1211, 2002.
Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013

316

KUCH, H. M.; BALLSCHMITER, K. Determination of

endocrine-disrupting phenolic compounds and estrogens
in surface and drinking water by HRGC- (NCI)-MS in
the picogram per liter range. Environmental Science
and Technology, v. 35, n. 15, p. 3201-3206, 2001.
LEE, H. B.; PEART, T. E.; SVOBODA, M. L.
Determination of endocrine-disrupting phenols, acidic
pharmaceuticals, and personal-care products in sewage by
solid-phase extraction and gas chromatography - mass
spectrometry. Journal of Chromatography A, v. 1094,
n. 1-2, p. 122-129, 2005.
LOPES, L. G.; MARCHI, M. R. R.; SOUZA, J. B. G.;
MOURA, J. A. Hormnios estrognicos no ambiente e
eficincia das tecnologias de tratamento para remoo em
gua e esgoto. Revista Brasileira de Recursos
Hdricos, v. 13, n. 4, p. 123-131, 2008.
MAGUIRE, R. J. Review of the persistence of nonylphenol
and ethoxylathes in aquatic environments. Water Quality
Research Journal of Canada, v. 34, n. 1, p. 37-78, 1999.
MARA, D. D. Water, sanitation and hygiene for the health
of developing nations. Public Health, v. 117, n. 6,
p. 452-456, 2003.
MATTHIESSEN, P.; GIBBS, P. E. Critical appraisal of
the evidence for tributyltin-mediated endocrine
disruption in molluscs. Environmental Toxicology
Chemistry, v. 17, n. 1, p. 37-43, 1998.
MEYER, A.; SARCINELLI, P.; MOREIRA, J. C. Estaro
alguns grupos populacionais brasileiros sujeitos ao dos
disruptores endcrinos? Cadernos de Sade Pblica,
v. 15, n. 4, p. 845-850, 1999.
MORAES, N. V.; GRANDO, M. D.; VALERIO, D. A.
R.; OLIVEIRA, D. P. Exposio ambiental a
desreguladores endcrinos: alteraes na homeostase dos
hormnios esteroidais e tireoideanos. Revista Brasileira
de Toxicologia, v. 21, n. 1, p. 1-8, 2008.
NELSON, J.; BISHAY, F.; VAN ROODSELAAR, A.;
IKONOMOU, M.; LAW, F. C. P. The use of in vitro
bioassays to quantify endocrine disrupting chemicals in
municipal wastewater treatment plant effluents. Science
of the Total Environment, v. 374, n. 1, p. 80-90, 2007.
NICHOLS, K. M.; SNYDER, E. M.; SNYDER, A.;
PIERENS, S. L.; MILES-RICHARDSON, S. R.;
GIESY, J. P. Effects of nonylphenol ethoxylate on
reproductive output and bioindicators of environmental
estrogen exposure in fathead minnows, Pimephales
Environmental
Toxicology
and
promelas.
Chemistry, v. 20, n. 3, p. 510-522, 2001.
OLEA-SERRANO, N.; FERNNDEZ-CABRERA, M.;
PULGAR-ENCINAS, R.; OLEA-SERRANO, F.
Produtos qumicos como desreguladores endcrinos:
substncias danosas e como devem ser testadas. Cadernos
de Sade Pblica, v. 18, n. 2, p. 489-494, 2002.

Acta Scientiarum. Technology

Ferreira

PETROVIC, M.; ELJARRAT, E.; LOPES DE ALDA, M.

J.; BARCEL, D. Recent advances in the mass
spectrometric analysis related to endocrine disrupting
compounds in aquatic environmental samples. Journal of
Chromatography A, v. 974, n. 1-2, p. 23-51, 2002.
RODRIGUEZ-MOZAZ, S.; ALDA, M. J. L.;
BARCELOS, D. Monitoring of estrogens, pesticides and
bisphenol A in natural waters and drinking water
treatment plants by solid-phase extraction-liquid
chromatography-mass
spectrometry.
Journal
of
Chromatography A, v. 1045, n. 1-2, p. 85-92, 2004.
SCHWAIGER, J.; SPIESER, O. H.; BAUER, C.;
FERLING, H.; MALLOW, U.; KALBFUS, W.;
NEGELE, R. D. Chronic toxicity of nonylphenol and
ethinylestradiol: haematological and histopathological
effects in juvenile Common carp (Cyprinus carpio).
Aquatic Toxicology, v. 51, n. 1, p. 69-78, 2000.
SUMPTER, J. P.; JOHNSON, A. C. Lessons learned from
endocrine disruption and their application to other issues
concerning trace organics in the aquatic environment.
Environmental Science and Technology, v. 39, n. 12,
p. 4321-4332, 2005.
STUMPF, M.; TERNS, T. A.; WILKEN, R.;
RODRIGUES, S. V.; BAUMANN, W. Polar drug
residues in sewage and natural water in the State of Rio de
Janeiro, Brazil. The Science of the Total
Environment, v. 225, n. 1-2, p. 135-141, 1999.
TAPIERO, H.; NGUYEN-BA, G.; TEW, K. D.
Estrogens and environmental estrogens. Biomedicine
and Pharmacotherapy, v. 56, n. 1, p. 36-44, 2002.
TSUDA, T.; SUGA, K.; KANEDA, E.; OHSUGA, M.
Determination of 4-nonylphenol monoethoxylate,
nonylphenol diethoxylate and other alkylphenols in fish
and shellfish by high-performance liquid chromatography
with
fuorescence
detection.
Journal
of
Chromatography B, v. 746, n. 2, p. 305-309, 2000.
WAISSMANN, W. Vigilncia sanitria e desreguladores
endcrinos. Cadernos de Sade Pblica, v. 18, n. 2,
p. 511-517, 2002.
WANG, Y.; HU, W.; CAO, Z.; FU, X.; ZHU, T.
Occurrence of endocrine-disrupting compounds in
reclaimed water from Tianjin, China. Analytical
and Bionalytical Chemistry, v. 383, n. 5, p. 857-863,
2005.

Received on July 9, 2010.

Accepted on June 29, 2011.

License information: This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.

Maring, v. 35, n. 2, p. 307-316, Apr.-June, 2013